I am trying to determine the reaction pathway for a key enzyme and drug target, thymidylate synthase (TS), that provides the sole de novo pathway for the biosynthesis of thymidine 5'-monophosphate (dTMP). As an essential enzyme for DNA synthesis, TS is found in almost all living species and many viruses. Thus, TS is an important drug target for the development of anti-cancer, anti-parasitic, anti-fungal, and anti-viral agents. TS uses methylene tetrahydrofolate as the carbon source for methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) in the reaction. A species that offers great advantage in access to mutagenesis, and for which a wildtype crystal structure has been determined at UCSF, is Lactobacillus casei (LCTS). Many kinetic studies on LCTS and mutants of the protein have been done in Dr. Santi's lab at UCSF. During the past year I have determined the crystal structures of three wildtype complexes of LCTS with the folate analog CB3717 and with the normal substrates methylene tetrahydrofolate and dUMP. The active site structure for one of these complexes reveals the conversion of substrates to the products dihydrofolate and dTMP within the crystal. The results of these research efforts are described in a recent publication. I am also completing high resolution refinements on binary and ternary complexes of two poorly active LCTS mutants (E60Q, R218K) with CB3717, dUMP, and FdUMP. This study suggests a structural basis for the relative inactivity of the mutant enzyme and a manuscript is being prepared. The graphics computers and software at the Computer Graphics Laboratory have been extremely helpful in these studies. With them I have been able to compare directly the active sites of the wildtype and mutant LCTS ternary complexes in order to deduce crucial changes in important water molecule positions and amino acid side-chain orientations that may affect enzyme activity.